A multi-level mass spectrometry pipeline for the analysis of whole proteoforms and their complexes

蛋白质组学是一种非常有价值的工具，可以用来阐明支持本病发生的分子机制。 疾病。蛋白质组的现代视野包含了“蛋白质形式”的广泛复杂性，或 蛋白质形式，由特定的遗传和化学修饰产生，可以改变和调节 蛋白质生物活性。在生物医学研究中，在三个不同的水平上分析蛋白质形式是至关重要的：第一， 一级结构的确定，包括修饰的身份和位置；第二，数量 或大量的这些修饰；最后，蛋白质形式组件的特征。这些 蛋白质复合体通过弱相互作用结合在一起，是真正的生物活性分子。 在活体内工作的机器。目前，蛋白质形式只能在全球范围内使用 质谱仪(MS)。这一新领域被称为自上而下的蛋白质组学(TDP)，但尚未得到发展 成为下一代测序甚至基于多肽的猎枪级别的可扩展解决方案 蛋白质组学，因为需要先进的技术来精确测量大的蛋白质形式。为了这个 原因是，TDP的研究通常局限于蛋白质形式&lt；30 kDa，它代表了不到一半的 哺乳动物蛋白质组。此外，这些研究通常是在还原、变性条件下进行的 促进分析前工作流程的条件，从而使蛋白质形式的更高阶结构丢失 以及半胱氨酸连接的翻译后修饰(PTM)。幸运的是，当代的 结构生物学家可以在“天然”状态下用MS分析多种蛋白质形式的复合体(MPC) 保持非共价相互作用的条件，尽管这项技术与高- 吞吐量研究，可用于发现新的生物学见解。在这里，我们提出了一个新的 TDP管道，扩展了可在发现模式下测量的蛋白质形式的质量范围，还 将变性MS和天然MS相结合来测量MPC。我们将首先结合创新分离 技术和气相化学(例如，新的离子碎裂技术和离子-离子反应) 为了改进高达100 kDa的蛋白质形式的检测和测序，并允许表征 被忽视的PTMS。然后，这些技术将被应用于蛋白质形式的定量&>30 kDa 在大规模的发现研究中(识别健康和健康人群之间的蛋白质形式表达差异 疾病状态)和有针对性的实验(以监测感兴趣的蛋白质形式的子集)。最后， 利用变性TDP收集单一蛋白质组分的定性和定量信息 实验将用于促进高通量本机中MPC的自动化表征 攻读硕士学位。这种多层次、以蛋白质形式为中心的全球蛋白质组分析方法将提供 研究人员拥有原本无法获取的信息，为制定小说打开了新的可能性 医疗和发现诊断疾病生物标记物的假说。

Proteomics is an invaluable tool for elucidating the molecular mechanisms that underpin the onset of disease. The modern vision of the proteome encompasses the broad complexity of “protein forms”, or proteoforms, created by specific sets of genetic and chemical modifications that can alter and regulate protein bioactivity. In biomedical research it is pivotal to analyze proteoforms at three distinct levels: first, determination of primary structure, including identity and position of modifications; second, the quantity or abundance of these modifications; and finally, characterization of proteoform assemblies. These protein complexes are held together by weak interactions and are the actual bioactive molecular machines that function in vivo. Currently, proteoforms can only be characterized at the global scale using mass spectrometry (MS). Known as top-down proteomics (TDP), this nascent field has not yet developed into a scalable solution on the level of next generation sequencing or even peptide-based shotgun proteomics, due to the advanced technology required to precisely measure large proteoforms. For this reason, TDP studies are typically limited to proteoforms <30 kDa, which represent less than half of the mammalian proteome. Furthermore, these studies are most often conducted under reducing, denaturing conditions to facilitate preanalytical workflows, so that higher order structure of proteoforms is lost together with cysteine-linked post-translational modifications (PTMs). Fortunately, contemporary structural biologists can analyze multi-proteoform complexes (MPCs) by MS under “native-like” conditions that preserve non-covalent interactions, although this technology is incompatible with high- throughput studies that can be leveraged to discover novel biological insights. Here, we propose a new TDP pipeline that extends the mass range of proteoforms measurable in discovery mode, and also integrates denaturing and native MS to measure MPCs. We will first combine innovative separation techniques and gas-phase chemistries (e.g., novel ion fragmentation techniques and ion-ion reactions) to improve the detection and sequencing of proteoforms up to 100 kDa, and allow the characterization of neglected PTMs. Then, these technologies will be applied to the quantification of proteoforms >30 kDa both in large-scale discovery studies (to identify proteoform expression variations between healthy and disease states) and in targeted experiments (to monitor subsets of proteoforms of interest). Finally, the qualitative and quantitative information collected on single proteoforms through denaturing TDP experiments will be used to facilitate the automatized characterization of MPCs in high-throughput native MS studies. This multi-level, proteoform-centric approach to the global analysis of proteomes will provide researchers with otherwise inaccessible information, opening new possibilities to formulate novel hypotheses for medical treatment and for the discovery of diagnostic disease biomarkers.